Clothing made from cellulosic fabrics such as cotton, linen, hemp, ramie, cupro, lyocell, newcell, rayon, polynosics, are very popular. Of particular interest are clothing items such as jeans made from indigo-dyed denim fabrics made of cotton or cotton blends. Such clothing items are typically sewn from sized and cut cloth and tend to be stiff due to the presence of sizing compositions. In other cases the fibers or rolls of fabric are treated with enzymes prior to sewing the final garment. After a period of wear, the clothing items can develop a certain degree of softness, an overall reduction of shade as well as localized areas of color variation. Additionally, after repeated washing the garment continues to provide a more comfortable fit, a softer feel and a worn appearance. In recent years such comfort, feel and appearance have become increasingly popular.
The most widespread methods for producing this comfort, feel and look involve washing of clothing items with cellulases in large washing machines with pumice stones or other abrasives. The pumice helps soften the fabric and helps to provide the faded surface similar to that produced by the extended wear of the fabric. However, the use of pumice has some disadvantages. For example, the pumice must be manually removed from processed clothing items because it tends to accumulate in pockets, on interior surfaces, in creases, and in folds. Also, the pumice stones can cause overload damage to electric motors of stone washing machines, and clog machine drainage passages and drain lines. These processing and equipment problems can add significantly to the cost of doing business and to the purchase price of the goods.
In view of the problems of using pumice, alternative methods to using pumice or other abrasives in the stone washing process have been sought. One alternative involves the use of enzyme treatments which break down the cellulose in fabrics (Geller U.S. Pat. No. 4,951,366; Olson U.S. Pat. Nos. 4,832,864, 4,912,056, Olson et al. U.S. Pat. Nos. 5,006,126, 5,122,159 and 5,213,581, Christner et al. U.S. Pat. No. 4,943,530, Boegh et al. U.S. Pat. No. 4,738,682). Methods for treating cellulose containing fabrics with hydrolytic enzymes, such as cellulases, are known in the art to improve the softness or feel of such fabrics (Novo Brochure Cellulase SP 227; Novo Brochure Celluzyme; Murata U.S. Pat. No. 4,443,355; Parslow U.S. Pat. No. 4,661,289; Tai U.S. Pat. No. 4,479,881; Barbesgaard U.S. Pat. No. 4,435,307; Browning UK Patent No. 1,368,599).
Cellulases are known in the art as enzyme systems that hydrolyze cellulose (.beta.-1,4-glucan linkages), thereby resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulase compositions are comprised of several different enzyme components, including those identified as exocellobiohydrolases, endoglucanases, and .beta.-glucosidases. Moreover, these classes of enzymes can be further separated into individual isoenzymes.
The complete cellulase system is required to efficiently convert crystalline cellulose to glucose. Generally, if total hydrolysis of a cellulose substrate is needed, the cellulase mixture should contain .beta.-glucosidases and cellobiohydrolases, as well as endoglucanases. Endoglucanases catalyze random hydrolysis of .beta.-1,4-glycosidic bonds between glucose units of cellulose polymers. Such components hydrolyze soluble cellulose derivatives such as carboxymethylcellulose, thereby reducing the viscosity of such solutions. Such enzyme components act on internal regions of the polymer, resulting in a rapid decrease in average polymer chain length together with a slow increase in the number of reducing ends. The rapid decrease in average chain length of the cellulose polymer is evidenced by the decrease in viscosity of a cellulose solution.
The substrate specificity and mode of action of the different cellulases varies among strains of organisms that produce cellulases. For example, the currently accepted mechanism of cellulase action in cellulase from the fungus Trichoderma reesei is that endoglucanase activity first break internal .beta.-1,4-glucosidic bonds in regions of low crystallinity of the cellulose (Ruohnen L., et al. In: "Proceedings of the Second Tricel Symposium on Trichoderma Reesei Cellulases and Other Hydrolases", (ed. by P. Sudminen and T. Reinkainen.,) Foundation for Biotechnology and Industrial Fermentation Research 8; (1993):87-96) The cellobiohydrolase activity binds preferentially to the crystalline regions of the non-reducing end of the cellulose to release cellobiose as the primary product. .beta.-Glucosidase or cellobiase activities then act on cellooligosaccharides, e.g., cellobiose, to give glucose as the sole product.
Cellulases are produced in fungi, bacteria, and other microbes. Fungi typically produce a complete cellulase system capable of degrading crystalline forms of cellulose. For example, Trichoderma reesei produces and secreates all of the enzyme activities needed for efficient breakdown of crystalline cellulose, namely endo-1,4-.beta.-D-glucanases, cellobiohydrolases (exo-1,4-.beta.-D-glucanases), and 1,4-.beta.-D-glucanases, or .beta.-glucosidases. Fungal cellulases have an added advantage in that cellulases in fungi can readily be produced in large quantities via fermentation procedures.
Cellulases, or the components thereof, are known in the art to be useful in a variety of industrial textile applications in addition to the stone washing process. For example, cellulases are used in detergent compositions, either for the purpose of enhancing the cleaning ability of the composition or as a softening agent. When so used, the cellulase will degrade a portion of the cellulosic material, e.g., cotton fabric, in the wash, which facilitates the cleaning and/or softening of the fabric. The endoglucanase components of fungal cellulases have also been used for the purposes of enhancing the cleaning ability of detergent compositions, for use as a softening agent, and for use in improving the feel of cotton fabrics, and the like. However, there is a problem with using the cellulase derived from Trichoderma spp. and especially Trichoderma longibrachiatum in detergent compositions. Generally, such components have their highest activity at acid pHs whereas most laundry detergent compositions are formulated for use at neutral or alkaline conditions.
Other textile applications in which cellulases have been used include softening (Browning, UK Patent No. 1,368,599, Parslow, U.S. Pat. No. 4,661,289, Tai U.S. Pat. No. 4,479,881 and Barbesgaard, U.S. Pat. No. 4,435,307), defibrillation (Gintis, D. Mead, E. J., Textile Research Journal, 29, 1959; Cooke, W. D., Journal Of The Textile Research Institute, 74, 3, 1983; Boegh, European Patent Application No. 0 220 016). Cellulases have also been used in combination with a polymeric agent in a process for providing localized variation in the color density of fibers. (WO/94/19528 and WP/94/1529).
Cellulases are classified in the garment and textile industry according to their pH range of operation. Acid cellulases typically have their peak activity at pH values of about 4.0 to 5.5 and less, neutral cellulases at about pH 5.5 to 7.5, and alkaline cellulases at about pH 7.5 to 11.0. Some enzyme compositions may have broader ranges of operation. For example, the neutral/alkaline cellulases may operate at acid, neutral and alkaline pH's at between about 40.degree. C. to 60.degree. C.
Acid, neutral and alkaline cellulases are typically used in the "stone wash" treatment of denim jeans, with or without surfactants, buffers, detergents, anti-redeposition agents, softening agents, pumice stones or other abrasives, bleaching agents, such as optical bleaching agents, enzymes, or other means.
If the cellulase composition is not formulated and/or pre-buffered then for acid cellulases, the pH is typically adjusted to between pH 4.5-5.5, with for example, a sodium citrate and citric acid buffer, and for neutral or alkaline cellulases between 5.5-7.5 with, for example, a monosodium and disodium phosphate buffer. Neutral and alkaline cellulases are typically used as additives to laundry detergents where the pH of operation may range from about pH 7.0 to 11.5. In stone wash applications typical acid cellulases generally provide greater backstaining or redeposition of the indigo dye and greater strength loss of the fabric while the typical neutral and alkaline cellulases generally provide less abrasion, lower backstaining or redeposition and less strength loss of the fabric.
The neutral/alkaline cellulases are the most preferred type of cellulases for the stonewash industry because they cause lower levels of backstaining or redeposition and lower strength loss than acid cellulases (ie, from Trichoderma spp.). Furthermore, neutral/alkaline cellulases, unlike their acid counterparts, operate at a much wider pH range and are able to maintain better relative wash performance within a wider pH range (pH 5.0-pH 8.0) in the stone washing industry. Therefore, neutral/alkaline cellulases provide several advantages. First, the incoming feed water in wet processing facilities is typically within this pH range lessening the need for as precise pH control as compared to acid cellulases. This makes the stonewashing process more tolerant to operator pH error or neglect leaving the overall procedure more forgiving than procedures using acid cellulases. Secondly, it is known that denim fabrics are alkaline in nature owing to the fact that the dyeing process utilities caustic soda. Simply washing denim releases this caustic into the wash water and the pH of the wash water generally rises. The alkalinity may overcome the bath buffers, but the effect of increased pH is less severe on neutral/alkaline cellulases compared to acid cellulases because neutral/alkaline cellulases operate not only at higher pH, but also over a wider pH range.
The wide spectrum of industrial uses for cellulases or the components of cellulases, especially alkaline and/or neutral cellulases, establishes a clear need for cellulases that are operative at neutral and/or alkaline pH. The present invention provides a procedure for producing neutral/alkaline cellulases having enzymatic activity at neutral and/or alkaline pH's and compositions comprising the same.